SUMMARY WHIRLY1 is a chloroplast‐nucleus located DNA/RNA‐binding protein with functions in development and stress tolerance. By overexpression of HvWHIRLY1 in barley, one line with a 10‐fold and two lines with a 50‐fold accumulation of the protein were obtained. In these lines, the relative abundance of the nuclear form exceeded that of the chloroplast form. Growth of the plants was shown to be compromised in a WHIRLY1 abundance‐dependent manner. Over‐accumulation of WHIRLY1 in chloroplasts had neither an evident impact on nucleoid morphology nor on the composition of the photosynthetic apparatus. Nevertheless, oeW1 plants were found to be compromised in the light reactions of photosynthesis as well as in carbon fixation. The reduction in growth and photosynthesis was shown to be accompanied by a decrease in the levels of cytokinins and an increase in the level of jasmonic acid. Gene expression analyses revealed that in nonstress conditions the oeW1 plants had enhanced levels of pathogen response (PR) gene expression indicating activation of constitutive defense. During growth in continuous light of high irradiance PR gene expression increased indicating that under stress conditions oeW1 are capable to further enhance defense. Significance Statement With regard to its dual localization in chloroplasts and nucleus WHIRLY1 is an excellent communicator in retrograde signaling. Previous studies with barley showed that a knockdown of WHIRLY1 compromises acclimation to environment. Here we show that overexpression of WHIRLY1 in barley, leading to an over‐accumulation of the protein in both chloroplasts and nucleus, improves stress resistance whereas it has a negative impact on photosynthesis and growth.